PROJECT SUMMARY This proposal describes a five-year career development program to prepare the candidate, Dr. Sang-Ging Ong, for a career as an independent investigator. This program will expand Dr. Ong's scientific background in cardiovascular research by providing additional technical training and expertise in mitochondria biology and stem cell biology, areas in which Dr. Ong has already made significant achievements. The mentor is Dr. Joseph Wu, a Professor of Medicine/Cardiology and Director of the Stanford Cardiovascular Institute at Stanford University. The proposed mentor is a physician scientist with significant expertise in stem cell biology and is an expert in cardiovascular disease modeling. The K99 phase will consist of structured mentorship by the primary mentor, complementary meetings with the advisory committee, formal coursework, a provocative research project, and a program of career transition. Diabetes is at epidemic proportions with 300 million people expected to suffer from diabetes by 2025. Cardiovascular disease is the major cause of death among these patients of which the major contributing factor is coronary artery disease (CAD). However, diabetic patients also suffer from diabetic cardiomyopathy (DCM) independent of the vascular effects of hypertension or CAD. The mechanisms underlying DCM are unclear, and there are currently no specific effective treatments for it. In all cells, including those of heart muscle, the autophagy/lysosome system provides proteolytic mechanisms to regulate protein turnover and degradation. Mitophagy is an autophagic process that specifically removes damaged mitochondria and may be crucial for the proper maintenance of cardiac function when in excess nutrient. The role of mitophagy in the diabetic heart is currently unknown, and in this proposal, Dr. Ong intends to understand the importance of mitophagy in DCM, and explore the underlying mechanisms that regulate mitophagy which may help in translational science. Combining Dr. Ong's expertise in mitochondrial biology with his skills in stem cell biology and genomics/proteomics biology that he is developing while working with Dr. Wu puts him in a unique position to be able to study the importance of mitophagy in human cardiac cells and the molecular mechanisms pertaining to it in ways that have not been done before. Dr. Ong has generated preliminary data demonstrating that mitophagy is impaired in human induced pluripotent-stem cells-derived cardiomyocytes (iPSC-CMs) subjected to hyperglycemia although there is a heterogeneous response. Dr. Ong will seek to conclusively prove that impaired mitophagy increases the susceptibility of iPSC-CMs to hyperglycemic damage, and to potentially identify a molecular signature of mitophagy which may be useful in the future for predicting response to excess glucose (Aim 1). Dr. Ong's preliminary results have also revealed an interesting phenomenon in that cells resistant to hyperglycemic stress are associated with an activation of non-canonical mitophagy despite impaired canonical autophagy. Hence Dr. Ong will investigate the mechanistic basis of impaired mitophagy and also the potential regulation of mitophagy by mild ROS (Aim 2). This is due to Dr. Ong's observation that treatment of iPSC-CMs with antioxidants worsen the detrimental effects of hyperglycemia, indicating an adaptive role of ROS (mitohormesis). In the final aim, Dr. Ong will obtain additional iPSC lines derived from diabetic patients with and without DCM, as well as introducing known diabetes susceptibility genes into isogenic healthy lines. These groups of lines will provide Dr. Ong a proof-of-principle platform for drug screening which shall be based on two formats: (i) testing for currently available anti-diabetic drugs as some drugs have been associated with adverse cardiac outcomes, and (ii) testing for mitophagy inducers which may protect against hyperglycemic damage which is based on my earlier findings (Aim 3). The implications of this work will be the identification of proper mitochondrial homeostasis as a requirement for prevention of DCM which may involve non-canonical mitophagy, highlight the importance of oxidative stress as mitohormetic molecules, and provide a unique human cardiac cells- based platform for drug testing. Completing these studies in a strong mentored environment will lay the foundation for Dr. Ong's transition to his own independent research program.